Understanding how genes are turned on and off is critical to understanding normal eukaryotic development and increases the probability of preventing or treating developmental anomalies and diseases. In Paramecium many genes code for large proteins, the i-antigens, that coat the surface of each cell. A cell is capable of producing any of about a dozen such proteins, but typically only one is produced because only one gene is transcribed. Different stocks show different arrays and frequencies with environment, cytoplasmic factors, and multiple genes important in the pattern. The A and G i-antigen genes in stocks 51 and 172 of P. tetraurelia are the focus of this study. These stocks show markedly different patterns of expression of the two genes. Specifically addressed is whether the genes or their flanking regions, likely important in control of expression, are structurally different. Techniques of gene cloning and standard molecular genetics techniques will be used to clone, identify and compare the genes. Whole genome DNA from the two stocks will be compared initially and then selection and characterization of cloned A and G genes from the stocks will begin. The long term goal at a more sophisticated level, including nucleotide sequencing of part or all of the structural regions and appropriate flanking areas. Such a detailed structural analysis will address the question of why the patterns of expression of these genes are so different in the two stocks. A biotinylated probe assay will be adapted for use with this system to avoid using radioactive probes while working with undergraduate students. Libraries of cloned DNA of stock 51, available from John Preer, Indiana University, and one or more newly prepared libraries of cloned DNA of stock 172 will be sources of clones of the A and G genes. Already characterized gene probe will be used when possible, and new ones selected as required.